Glucose dehydrogenase and production thereof

ABSTRACT

The invention relates to novel PQQ-dependent soluble glucose dehydrogenases (sPQQGDH) from  Acinetobacter  and to a process for their preparation by overexpression in suitable microbial expression systems.

The invention relates to novel PQQ-dependent soluble glucosedehydrogenases (sPQQGDH) from Acinetobacter and to a process for theirpreparation by overexpression in suitable microbial expression systems.

Patients suffering from diabetes must measure their blood glucoseregularly. Measurement of glucose likewise plays an important part infermentation processes. In many cases, glucose is determinedenzymatically. For this purpose, either glucose oxidase, or, in rarecases, also glucose-6-phosphate dehydrogenase are employed. The methodsbased on glucose oxidase have the disadvantage that the enzyme transfersthe electrons not just to the added mediators but also to the oxygenpresent. The result of the measurement is therefore dependent on theoxygen partial pressure. Efforts have been made for some time to replacethe present enzymes by sPQQ-dependent glucose dehydrogenases. PQQ standsfor pyrroloquinoline quinone. PQQ is the prosthetic group of glucosedehydrogenase (GDH). It transfers redox equivalents. The advantage ofthese enzymes is that the measurement is independent of the oxygenpartial pressure and a more accurate measurement is possible. Inaddition, measurement in smaller sample volumes is possible through useof these enzymes.

Two different PQQGDHs are to be found in the literature. One form ismembrane-bound (mPQQGDH) and is unsuitable for use in glucose sensors.The other form is soluble (sPQQGDH) and has been found variously instrains of the genus Acinetobacter (Biosci. Biotech. Biochem. 59 (8),pp. 1548-1555, 1995). The enzyme is a homodimer and has a molecularweight of 50 kDa. The soluble and the membrane-bound PQQGDH have nosequence homology and are different immunologically and in terms oftheir kinetics (Cleton-Jansen et al., 172 (11), pp. 6308-6315, J.Bacteriol. 1990) (Matsushita et al., Biochemistry 28 (15), pp.6276-6280, 1989).

The sPQQGDH from Acinetobacter has been known for some time. All authorsuse the strains LMD 79.41 (Kojima et al., Biotechnology Letters, 22, pp.1343-1347, 2000), NCIMB 11517 or JCM 6841 (both: US 2001021523).

The DNA sequences of the sPQQGDH of these strains are deposited in theGenbank under the access numbers X15871 (LMD 79.41), E28183 (JCM 6841)and E28182 (NCIMB 11517).

Many attempts have been made to alter the properties of these sPQQGDH bymodifying the gene sequences. (EP 1 167 519 A1, EP 1 176 202 A1, Sodeund Kojima, Biotech. Letters, Vol. 19, (11) pp. 1073-1077, 1997). Theaim in these cases was to improve the substrate specificity and thethermal stability of the enzyme. EP 1 167 519 A 1 describes thereplacement of individual amino acids of the sPQQGDH from AcinetobacterLMD 79.41 in order to obtain increased thermal stability. Alterationsare made in amino acids 209, 210, 231, 420 and 421. WO 02/072839 A1describes further alterations in the amino acid sequence in order toachieve increased thermal stabilities and improvements in the watersolubility. Replacements were made in positions 167, 231, 340, 415 and418. The numbering is based in both cases on that used in EP 1 167 519.When comparing the enzyme from Acinetobacter LMD 79.41 with the novelsPQQGDH according to the invention and that from Acinetobacter JCM 6841it is necessary also to take account of the fact that the twolast-mentioned enzymes are two amino acids longer than that from LMD79.41. The mature enzyme and the signal peptide are considered in thisconnection.

WO 02/34919 A1 describes replacement of individual amino acids with theaim of reducing the affinity of the enzyme for maltose. The thermalstability of the enzyme is unaffected.

Enzymes are normally prepared by heterologous expression thereof in E.coli. The problem arising on expression of sPQQGDH is that although E.coli can synthesise the enzyme, it cannot synthesise cofactor PQQ.Although, according to Sode et al. (J. Biotechnology, 49, 239-243, 1996)expression of the apoenzyme mPQQGDH is possible in the case of themembrane-bound glucose dehydrogenase, it is unstable and is broken downagain during culturing of the cells. Matsushita et al. (Biosci.Biotechnol. Biochem. 59, 1548-1555, 1995) postulate a conformationalchange in the enzyme occurring on binding of the cofactor and protectingthe holoenzyme from tryptic digestion.

For this reason, all research groups and manufacturers have made effortsto prepare active and stable enzyme in such a way that either PQQ isadded during culturing of the cells, or the expression takes place instrains themselves able to form PQQ. Sode et al. (J. Biotechnology, 49,239-243, 1996) succeeded in coexpressing mPQQGDH together with the genesfor PQQ synthesis in E. coli and achieved about 1500 U/I at an OD of 4.Addition of PQQ during culturing of the cells achieves 1100 U/I.However, the system has the problem that the cells do not reach highoptical densities and die during enzyme production (Kojima et al.,Biotechnology Letters, 22, 1343-1347, 2000). The authors in thepreviously cited publication describe the synthesis of active sPQQGDH inKlebsiella pneumoniae. This organism is genetically available and hasthe ability to synthesize PQQ. In this case, therefore, activities ofabout 14 000 U/I are reached, although additional PQQ feeding isnecessary. Yoshida et al. (Enzym. Microb. Technol. 30, pp. 312-318,2002) expressed soluble sPQQGDH in Pichia pastoris heterologously andthus achieved usable enzyme yields of up to 200 000 U/I. However, it isnecessary for this purpose to reach very high cell densities. Inaddition, the enzyme is glycosylated and must additionally be purifiedby a precipitation and two ion exchanges. Cost-effective preparation isimpeded in particular by the elaborate purification and theglycosylation.

The Toyobo Co. Ltd., Japan, describes in JP 09140378 the preparation ofsPQQGDH using Acinetobacter calcoaceticus. 60 U/I are reached afterthree purification steps. In order to increase the productivity of thesystem, the enzyme was subsequently expressed heterologously inPseudomonas putida, because this strain is able to synthesize PQQ.

Olsthoorn and Duine (Arch. Biochem. Biophys. 336 (1), 42-48, 1996)describe the batch culturing of an E. coli clone which expresses sPQQGDHin a 100 l fermenter. After the culturing, the enzyme is purified bythree column steps. The clone forms no PQQ; the cofactor is added onlyin the enzyme assay. The yields amount to 10 mg of pure protein per 1 ofgrown culture. The low cell density of the culture, the elaboratepurification process and the yield, however, make the process appearuneconomic.

DESCRIPTION OF THE INVENTION

During a screening, 12 strains of the species Acinetobactercalcoaceticus which form an sPQQGDH were found. Typing of the novelstrains by the Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) revealed 99.8% homology of the partial 16S rDNA sequence to theAcinetobacter calcoaceticus type strain. However, the observedutilization of L-malate and the inability to break down L-phenylacetateis unusual for the type strain This property is shown by all 12 strainsfound.

The sPQQGDHs formed by these strains differ from the previously knownenzymes in substantial properties. On the one hand, they have a thermalstability which is improved by comparison with the enzyme from the typestrain (see Example 3). The nucleotide sequences and the sequence of theamino acids are likewise different.

Sequence analysis of the 12 genes which code for the novel sPQQGDHsrevealed the following results (see FIG. 1):

The nucleotide sequences of all the newly found sPQQGDHs differdistinctly from those of the enzymes from Acinetobacter LMD 79.41 andAcinetobacter JCM 6841 and NCIMB 11517.

The amino acid sequences which can be derived from the respectivenucleotide sequences likewise differ from the previously knownsequences. The sPQQGDH from Acinetobacter LMD 79.41 has 478 amino acids,including the signal peptide, while those from the two other knownstrains Acinetobacter JCM 6841 and Acinetobacter NCIMB 11517, and thosefrom the sequences found here each have 480 amino acids, includingsignal peptide.

The amino acid sequences of the newly found sPQQGDHs differ in numerouspositions from those previously known. Surprisingly, an amino aciddifferent from that in all previously described enzymes was found at 12positions with all the genes newly described herein. In addition, atleast 75% of the genes newly described herein have an amino aciddifferent from that in the previously described genes at four positions.Moreover, in the enzymes newly described herein there are furtherexchanges, which occur singly or in some of the enzymes.

The specific replacements in the newly found sPQQGDH according to theinvention are of the following amino acids. The numbering of amino acidsis based on the numbering in the enzyme from the strain AcinetobacterJCM 6841, which comprises 480 amino acids, including signal peptide (seeFIG. 1):

All the sPQQGDHs which have at positions 21 an N and 41 an S and 47 an Land 121 a V or an A and 149 an A and 213 an S and 244 an I and 320 a Gand 391 an S and 452 an S and 474 an R and 480 a Q as amino acid areaccording to the invention.

Further according to the invention are all the sPQQGDHs in which thefollowing exchanges may occur in addition to the exchanges describedabove:

There may be at positions 16 an H, at 18 an L, at 20 an F, at 40 a G, at48 an I, at 61 an A, at 111 a T, at 154 a D, at 190 a D, at 293 an A, at311 an S, at 314 an A, at 324 an L, at 333 an M, at 339 an S, at 355 aG, at 366 a D, at 417 an N and at 418 an A.

It has surprisingly been found that the newly found enzymes are all morethermally stable than the wild-type enzyme. The thermal stability is anessential criterion for successful use of sPQQGDH as glucose sensor.Thus, the remaining activities found on incubation at 60° C. and 70° C.for one hour were all higher than with the enzyme from Acinetobacter LMD79.41. The remaining activity found at 60° C. with the sPQQGDHsaccording to the invention were between 51.5% and 12.5%. The wild typeshowed only 1.8% of its original activity in an identical approach.Between 26.7% and 8.8% of the initial activity were observed at 70° C.,whereas the wild-type enzyme had only 6.4% of the original activity.

Various attempts have been made in the past to increase the thermalstability of sPQQGDH by targeted and random exchange of amino acids.

However, EP 1 167 519 A1 claims the exchange of amino acids at positionsmarkedly different from those in the sPQQGDHs according to theinvention. The same applies to the exchanges described in WO 02/072839.This likewise applies to the exchanges described in WO 02/34919 A1. Theincreased thermal stability of the sPQQGDHs according to the inventionthus correlates with the exchange of a whole group of amino acids whoseinfluence on this property was not previously known.

The enzymes according to the invention can be prepared by cultivatingthe relevant strain in a suitable medium. It is possible to use for thispurpose the media described in the literature, such as nutrient broth(Difco 0003). After the cells have grown, they are harvested anddisrupted. The enzyme is purified as described below.

The sPQQGDHs from the wild type are preferably cloned into a suitablehost. Possibilities therefor are the usual genetically readily availableprokaryotes such as members of the genus Bacillus, Klebsiella,Pseudomonas, and E. coli. The enyzmes may furthermore also be expressedin eukaryotes such as members of the genera Pichia, Saccharomyces,Hansenula, Aspergillus or Kluyveromyces. The enzymes may also beexpressed in plants or animal cell lines.

It is possible to employ for the cloning standard methods such as PCRwith degenerate primers or hybridization of genomic libraries withsuitable probes. Expression is preferably carried out in E. coli. Theexpression is normally carried out in the strains BL21, DH5, HB101,JM101, RV308, TOPP, TOP10, XL-1 and derivatives thereof. The strainsW3110 and DH5 are preferably used. Conventional expression vectors canbe employed for this purpose. The vectors typically comprise an originof replication, an antibiotic resistance and a promoter sequence.Examples of vectors which can be employed are the following: pUC18/19,pBluescript, pTZ, pGEX, pPROEx, pcDNA3.1, YEp24, pBAC, pPICZ. Vectors ofthe pMAL, pET, pTrx, pCAL, pQE and pPROTet series are preferred.Expression vectors of the pASK-IBA 2 to pASK-IBA 7 series areparticularly preferably employed. Usual antibiotics for resistanceselection are, for example, ampicillin, kanamycin, tetracycline andchloramphenicol.

It is also possible to use expression systems which lead to the proteinbeing secreted into the medium.

The vectors can be transferred to the host cell by conventional methods.Examples employed for this purpose are: electroporation, protoplastfusion, chemical transformation.

Synthesis of the cloned sPQQGDHs is induced by adding an inducer. Theinducers employed for this purpose are those suitable for the chosenexpression system, such as, for example. IPTG, tryptophan, glucose andlactose. The inducer anhydrotetracycline is particularly preferablyused.

The recombinant cell lines are cultured in media suitable therefor.Conventional processes used for culturing prokaryotes and eukaryotes aresuitable for this purpose. The culturing can be carried out in suitablefermenters. The organisms are preferably cultivated in such a way thatvery high cell densities are reached. To do this it is necessary for thefeeding with a C source and an N source to be suitably controlled by astrategy such that no toxic metabolic products result (for overview:Schügerl et al. (editors) in: Bioreaction Engineering, pp. 374-390,Springer-Verlag, Berlin, 2000; Yee and Blanch, Bio/Technology, 10 (2),pp. 1550-1556, 1992).

A process suitable for the purposes of this invention is for examplethat of Riesenberg et al., Appl. Microbiol. Biotechnol, 34, pp. 77-82,1990.

Whereas the cultivation of the cells ideally takes place at 28-37° C.,the temperature is lowered to 10-28° C. for expression of the protein.It is preferably at 15-25° C. and particularly preferably at 20-22° C.

It has surprisingly been found that on use of the process according tothe invention distinctly higher yields of active enzyme are attainedthan previously described in the literature. When an E. coli clone iscultured in a batch culture it is possible in this way to obtain up to48 mg of pure protein from one litre of culture supernatant (seeExamples 5 and 6). Yoshida et al. (Enzym. Microbiol. Technol., 30, pp.312-318, 2002) attained 43 mg/l. However, elaborate purification of theenzyme is necessary, and it is glycosylated. Olsthoorne and Duine (Arch.Biochem. Biophys. 336 (1), pp. 42-48, 1996, report a yield of 10 mg/l.

If the cells having the enzymes according to the invention are culturedin fermentation with high cell density, it is in fact possible to reachmore than 220 mg of pure enzyme per 1 of culture liquid (Example 8).

Culturing of the cells is followed by harvesting and disruption thereofby suitable methods such as, for example, French press, addition ofdetergents or ultrasound. The protein solution is buffered and broughtto a slightly alkaline pH. The buffer is adjusted to a pH between 7 and9, and is preferably between 7.8 and 8.2.

Buffer substances which can be employed are the buffers customary inbiochemistry, such as Tris, MOPS or PIPES buffers, potassium phosphatebuffer; the concentration ought to be 5-100 mM, and is preferably in arange of 20-70 mM and particularly preferably 50 mM.

The sPQQGDH can now be purified very easily from the protein solution.To do this, the protein solution is either purified by a conventionalion exchange chromatography, or the ion exchange material is directlyadded to the protein solution and then separated from the remainder ofthe liquid on a suction funnel. Suitable ion exchangers are cationexchangers such as, for example, Lewatit resins CM-, S—, SM-Sepharose,CM-, SP-Sephadex, Amberlyst 15, Amberlite CG-50, Amberlite IR-120,carboxymethyl-, sulphoxyethyl-, oxycellulose, cellulose phosphate andCM-Toyopearl. CM-Toyopearl is preferably employed.

The protein is then eluted from the ion exchange material byconventional methods. An increasing NaCl gradient is employed for this,and the buffer used is that also employed previously for binding theprotein to the ion exchange material. The enzyme is normally eluted at aconcentration of about 200 mM NaCl.

The enzyme can then be purified further, and it is likewise possible toreduce the salt content by conventional methods such as dialysis andultrafiltration.

The preparation and purification of the sPQQGDH according to theinvention preferably takes place as apoenzyme, and the cofactor PQQ isadded only when the enzyme has been completely purified. Addition of thePQQ ideally takes place in conjunction with changing the buffer of theenzyme after purification on the ion exchanger.

The PQQ can be added before the buffer is changed or thereafter. It ispreferably added beforehand. The amount depends on the content of theprotein solution. From 0.1 to 5 mol of PQQ can be added per mole ofactive enzyme. It is ideal to add from 0.5 to 2 mol, particularlypreferably 2 mol, of PQQ per mole of active protein. However, it is alsopossible for the enzyme to be prepared and purified as holoenzyme. Forthis purpose the PQQ can be added during cell culturing, during celldisruption or prior to purification. A further possibility is for thesPQQGDHs according to the invention also be prepared as holoenzymes byheterologous expression thereof in organisms able to synthesise PQQ.These may be for example organisms from the genus Klebsiella orPseudomonas.

The novel sPQQGDHs are employed according to the invention for glucosemeasurement. They are particularly preferably employed in instrumentswhich can be used to measure blood glucose. It is also possible inaddition to employ the enzymes for glucose measurement for example infermentation processes.

If the sPQQGDHs according to the invention are employed for diagnosticpurposes, such a test kit typically includes a buffer, a mediator andsome units of enzymic activity. 0.5-10 U are typically employed, and 1-5U are preferably employed. Various formulations of the enzyme arepossible for this purpose. It can for example be freeze- or spray-driedand formulated as solution. Suitable electrodes may be carbon, gold orplatinum electrodes. The enzyme is normally immobilized on theelectrode. Crosslinking agents are normally used for this purpose butthe enzyme can also be encapsulated. Further possibilities forimmobilizing it are by means of a dialysis membrane, byphotocrosslinking, electrically conducting or redox polymers.Combinations of the abovementioned methods are also possible.

The enzymes are preferably applied as holoenzymes but they can also beemployed as apoenzymes, in which case the necessary PQQ is supplied in asecond layer. The novel sPQQGDHs are preferably immobilized on a carbonelectrode with glutaraldehyde and then treated with a reagent containingamines for complete reaction of excess glutaraldehyde.

EXAMPLES Example 1 Search for Novel Strains Which Produce sPQQGDH

Soil samples were suspended in saline (0.9% NaCl), and aliquots werestreaked onto agar plates which contained a nutrient medium withgluconate as sole carbon source. The medium had the followingcomposition: NaCl 5 g MgSO₄ 0.2 g NH₄H₂PO₄ 1 g K₂HPO₄ 1 g Sodiumgluconates 2 g Yeast extract 0.5 g Agar 15 g Dist. water 1000 ml pH 7.0

The plates were incubated at 30° C. for 24-48 hours.

Alternatively, Pseudomonas-Agar from Oxoid was also used. The incubationconditions were identical.

Selected grown colonies were isolated on glucose-eosin-methylene blueagar. This had the following composition: K₂HPO₄ 1 g Glucose 18 gPeptone 10 g Eosins 0.4 g Methylene blue 0.06 g Agar 16 g Dist. water.1000 ml pH 7.6

The plates were incubated at 30° C. for 24 hours. Positive clones can beidentified by being dark red and/or having a green lustre. Thesecolonies are again streaked on nutrient agar (Oxoid) in order to checktheir purity. The composition thereof was as follows: Meat extract 1 gYeast extract 2 g Peptone 5 g NaCl 5 g Agar 15 g Dist. water 1000 ml pH7.4

The plates were incubated at 30° C. for 48 hours and again isolated. Thepurified strains were subsequently cultured in 100 ml of liquid nutrientbroth (Oxoid) at 30° C. for 20 h; the medium had the followingcomposition: Meat extract 3 g Bakto peptone 5 g Glucose 1 g Dist. water1000 ml

Culturing was followed by harvesting of the cultures (4500×g, 40 min, 4°C.) and washing with saline (0.9% NaCl). The pellets were resuspended in5 ml of 50 mM KP buffer of pH 7.2 and disrupted with ultrasound. Theextract was again centrifuged at 10 000×g at 4° C. for 30 min, and theGDH activity in the cell-free supernatant was measured. It was possibleto isolate 26 strains with GDH activity on the screening medium withgluconate as sole carbon source. It was possible to isolate four strainsafter culturing on the Pseudomonas medium.

Example 2 Purification of the Enzymes

The strains whose sPQQGDHs were to be investigated were cultured in 81of NB medium (Oxoid), which contained 0.1% glucose at 30° C. for 20 h.The cells were harvested (4500×g, 40 min, 4° C.), washed with 0.9%saline and taken up in 150-200 ml of 10 mM MOPS of pH 8.0. The cellswere disrupted with ultrasound. The cell-free extract was loaded onto300 ml of TSK gel CM-Toyopearl 650M (Tosoh Corp.) and washed with 3-4column volumes of 10 mM MOPS of pH 8.0. Elution took place with a 0-0.3M NaCl gradient with a 3-4-fold column volume. Active fractions werepooled, transferred into a dialysis tube and concentrated by addition ofhigh-viscosity carboxylmethylcellulose as water absorber. Theconcentrated sample was resuspended with 3.5 volume of 10 mM K-MOPS ofpH 6.8 and again purified on the CM-Toyopearl column. Equilibration ofthe column and washing took place with 10 mM K-MOPS of pH 6.8, andelution was carried out with a 0-0.3 M NaCl gradient. The activefractions were pooled and concentrated as described above. They servedas starting material for determining the thermal stability.

Example 3 Test of Thermal Stability

The thermal stability was determined by incubating the purified enzymesat 50, 60 and 70° C. for 60 minutes. The incubation took place in 50 mMPipes at pH 6.5 in the presence of 1 mM CaCl₂, 0.1% Triton X-100, 0.1%BSA and 5 μM PQQ. After the incubation, the samples were cooled on iceand the remaining activity was determined. It was expressed as apercentage of the original activity. TABLE 1 Thermal stability of novelsPQQGDH from various new isolates Strain 50° C. 60° C. 70° C. KGN25 10014.3 13.1 KGN34 104.2 19.8 14.6 KGN80 94.3 22.6 18.9 KGN100 95.9 12.513.5 KG106 102.6 33.4 13.9 KG140 105.5 20.5 8.8 KOZ62 107.7 42.8 14.7KOZ65 103.8 34.6 19.2 PT15 100.0 25.4 16.3 PT16 85.0 51.5 14.9 PTN26120.7 27.5 22.4 PTN69 93.2 36.4 26.7 LMD79.41 wild type 95.6 1.8 6.4

Example 4 Cloning and Analysis of the Novel sPQQGDH Genes

For cloning the sPQQGDH genes from the novel strains described here itwas initially attempted to amplify the complete coding sequence ongenomic DNA with synthetic oligonucleotides in a PCR. The primers whichwere used for this and were derived from the published sPQQGDH sequence(X15871; LMD 79.41) did not, however, lead to PCR products, because thesequences of the sPQQGDHs according to the invention differ particularlygreatly from the wild type in the region 15 of the signal peptide andclose to the stop codon. For this reason, various primers from bothstrands of the wild-type sequence which were intended to lead on use ina PCR to amplification of fragments of the coding sequence were used. Itwas possible with some of these primer combinations to isolate suchfragments from the strains described in Example 3. Commercial kits wereused for all the PCR reactions, usually the PCR master kit from Roche,in accordance with the manufacturer's instructions. It was possible withthe primers

GDH-fwd P1 (5′-CCA GAT AAT CAA ATT TGG TTA AC-3′) and

GDH-rev P7 (5′-CAT CAC GAT AAC GGT TYT TGC-3′) to isolate fragmentsabout 1200 bp in size. The resulting DNA pieces were then sequenced. Aninverse PCR was carried out in order to obtain the complete sequence ofthe individual genes (Sambrook and Russell: Molecluar Cloning—ALaboratory Manual. CSHL Press (2001), p. 8.81). The primers employed forthis purpose in the present case were each positioned on the margin ofthe fragment in such a way that the still unknown part of the GDH geneis synthesized in a further PCR. Such a PCR reaction was carried out ongenomic DNA of the respective strain, which had been cut with therestriction endonucleases EcoRI and BglII and then recircularized by T4ligase. The primers used were

GDH-3Mid (5′-GGGATATGACCTACATTTGCTGGC-3′) and

GDH-5Mid (5′-TGTCCATCAGCRTCATTTACAAYCTCAG-3′), which initiated directedDNA synthesis respectively in the direction of the 3′ end (GDH-3Mid) and5′ end (GDH-5Mid) of the GDH gene. The amplicons obtained in this waycontained the as yet missing portions of the coding sequence, as emergedby cloning into the vector pCR2.1 and subsequent sequencing. The DNAsequences which were now completely available were used anew to developprimers which made it possible for the coding sequence from start codonto stop codon to be cloned directly. The primers were chosen in thiscase so that cloning into the vector pASK-IBA3 is possible in accordancewith IBA's instructions. For this purpose, a BsaI cleavage site ispositioned directly in front of the coding sequence (in the 5′-bindingprimer) and one was positioned directly thereafter (in the 3′-bindingprimer) so that directed ligation of the BsaI-cut PCR product into theBsaI open vector pASK-IBA3 is possible. The 5′ primer used was

GDH-U3(5′-TGGTAGGTCTCAAATGAATAAACATTTATTGGCTAAAATTAC-3′), and the 3′primers used were GDH-L3

(5′-ATGGTAGGTCTCAGCGCTCTGAGCTlTlATATGTAAACCTAATCAAAG-3′; for the GDHfrom clone PT15) and GDH-L4

(5′-ATGGTAGGTCTCAGCGCTCTGAGCTTATATGTAAATCTAATCAGAG-3′;

for all other clones). The resulting plasmid as were referred to aspA13-X. Instead of “X”, the strain number of the clone from which thegenomic DNA was isolated is inserted. For the purposes of comparison,the sPQQGDH gene from the wild-type strain LMD79.41 was cloned in thesame way. However, the primers used for this were derived from thepublished sequence. This plasmid was referred to as pAI3-wt.

The described plasmids were transferred into the E.coli strain DH5α(from Invitrogen) by chemical transformation. The bacterial strainsobtained in this way were referred to as DH5α::pAI3-X. The plasmids weretransferred in a similar way into a further E.coli strain, W3110 (ATCC27325). These strains were then referred to as W3110::pAI3-X.

Example 5 Recombinant Preparation of sPQQGDH with E. coli

The preculture was prepared as follows. 0.1 ml of a glycerol stock ofDH5α::pAI3-KOZ65 cells was added to 2 ml of LB medium (50 μg/mlampicillin) and shaken at 37° C. and 225 rpm overnight. For the mainculture, 1 l of TB medium (50 μg/ml ampicillin) was inoculated with thefully grown preculture. The culture was shaken at 37° C. and 225 rpmuntil an OD of about 1 was reached. The main culture was then inducedwith an anhydrotetracycline (AHT) stock solution. The inducer wasdissolved in DMF for this purpose. The final concentration of AHT in theculture was 0.2 μg/ml, and induction took place at 27° C. and 225 rpmfor 24 hours. The OD of the main culture was then 4.2.

The cells were harvested by centrifugation of the main culture at 3220×gand 4° C. for 15 min. The cell pellets were resuspended in 40 ml (=1/25of the total volume) in 75 mM Tris-HCl of pH 8.0, and disrupted using aFrench press. This is done by treating the complete cell suspension withthe French press twice. The lysate, which may be cloudy due to inclusionbodies and cell detritus, was centrifuged at 48 745×g and 4° C. for 10min. The protein content and the activity of the supernatant wereassayed. The protein content was 9.25 mg/ml, and the activity was 1.4kU/ml. Based on the original culture, 56 kU were obtained per 1 ofculture.

Example 6 Purification of the Recombinant sPQQGDH

11.2 ml of the supernatant from Example 5 were separated bychromatography on a cation exchanger (Toyopearl CM-650M, from TOSOHBIOSEP GmbH) at 4° C. An XK 50/20 column (from Amersham PharmaciaBiotech) with a column bed of about 130 ml is used for this purpose; theflow rate was 8 ml/min. The column was equilibrated with 10 columnvolume of 10 mM K-MOPS of pH 8.0+1 mM CaCl₂ after which the sample isloaded. The column was washed with 4 column volume of 10 mM K-MOPS of pH8.0+1 mM CaCl₂, and it was then eluted with a linear salt gradient from0 to 0.4 N NaCl in 10 mM K-MOPS of pH 8.0 (in each case including 1 mMCaCl₂), collecting the eluate in fractions. Regeneration of the columntakes place with 3 column volume of 1 N NaCl+1 mM CaCl_(2.)

The eluate fractions were assayed for activity in 96-well microtitreplates with flat base in order to find the active fractions. The coloursolution had the following composition:

0.2 mM PMS (phenazine methosulphate in H₂O)

+0.22 mM NTB (nitrotetrazolium blue in H₂O)

+3 μM PQQ Na salt (in DMSO)

in 20 mM Tris/HCl of pH 7.5 with 2% glucose

The assay was carried out as follows: in each case 90 μl of sample wereintroduced into a microtitre plate, and 110 μl of colour solution wereadded to each; the colour reaction can usually be observed after onlyone minute. On the basis of the results in the online UV chromatogramand in the activity assay, the active fractions are combined and thepool is concentrated where appropriate by ultrafiltration (30 000 MWCO).A protein determination and a quantitative activity assay follow (seeExample 9). TABLE 2 Purification of the sPQQGDH from E. coliDH5α::pAI3-KOZ65 Total Total Specific Vol. protein activity activityYield [ml] [mg] [U] [U/mg] [%] Sample loaded 11.2 103.6 15 680 151 100onto column Active fractions 4.45 13.50 15 085.5 1117 96

Only one active band was detected in an SDS gel and in a native gel. Theprotein was thus pure. Based on the original culture, 48.2 mg of pureprotein were obtained per 1 of culture.

Example 7 Preparation of Recombinant sPQQGDH From W3110 Strains andTesting of the Thermal Stability

For comparison of the thermal stability of recombinant sPQQGDH fromLMD79.41 (plasmid pAI3-wt) and the sPQQGDH from KOZ65 (plasmidpAI3-KOZ65), the strains W3110::pAI3-wt and W3110::pAI3-KOZ65 werecultured in 200 ml of TB medium with 100 μg/ml ampicillin. After thecultures had reached an OD₆00 of 3, the bacteria were centrifuged at4600 rpm for 10′, and the pellets were taken up in each case in 25 ml offresh TB medium with 100 μg/ml ampicillin. AHT was added to aconcentration of 2 μg/ml for induction, and the cells were shaken at 22°C. for 6 h. The cells were then pelleted anew, and the pellets werestored at −80° C. until processed further. The frozen cells wereresuspended in each case in 25 ml of MOPS buffer (10 mM MOPS pH 8, 2.5mM CaCl₂, 0.05% Triton X-100) and disrupted by ultrasound treatmentuntil the suspension became distinctly clearer. Cell residues wereremoved at 20 000 rpm and 4° C. for 30 min, and the supernatant waspurified on a Toyopearl CM-650 M column (20 ml bed volume). For thispurpose, after the sample loading the column was washed with 50 ml ofMOPS buffer (see above) and then bound protein was eluted with agradient from 0 to 0.6 mM NaCl in MOPS buffer. Fractions with GDHactivity were pooled, and the activity of the pool was determined (seeExample 9).

The thermal stability of both enzyme preparations was determined bydilution with 50 mM Pipes, pH 6.5 with 1 mM CaCl₂, 0.1% Triton X-100,0.1% BSA and 5 μM PQQ to adjust to a solution of 20 U/ml. Aliquots ofthis solution were incubated in parallel at 4° C., 50° C., 57° C. and64° C. for 60 min. The remaining activity is then found, based on thevalue at 4° C. as 100%, as follows (as triplicates; see Example 9):Strain 50° C. 57° C. 64° C. W3110::pAI3-wt 95.1 ± 2.9 20.7 ± 0.8 3.4 ±0.0 W3110::pAI3-KOZ65 95.1 ± 1.6 56.8 ± 3.6 8.7 ± 0.3

Example 8 Preparation by High Cell-Density Fermentation

The fermentation was carried out in a 10 litre steel fermenter (BIOSTATC) from Braun.

5 litres of (modified) Riesenberg medium were employed for this purposeKH₂PO₄ 13.3 g (NH4)₂HPO₄ 4.0 g Citric acid 1.7 g Magnesium sulphate ×7H₂O 1.2 g Thiamine 0.5 g Tryptone 1.2 g Yeast extract 2.4 g Traceelement solution 50 ml Dist. water ad 880 ml Glucose 5 g in 100 ml ofdist. water (sterilized separately) Ampicillin 100 mg (separatelydissolved in 20 ml of dist. water and sterilized by filtration) Traceelement solution Titriplex III 0.84 g Fe(III) citrate 6.00 g MnCl2 ×4H₂O 1.50 g ZnCl2 × 2H₂O 0.80 g H₃BO₃ 0.30 g Na₂MoO₄ × 2H₂O 0.25 g CoCl₂× 6H₂O 0.25 g CuCl₂ × 2H₂O 0.15 g Dist. water ad 1000 ml

The pH was adjusted after addition of all the medium ingredients to 6.80with 5 N NaOH.

The nutrient solution was inoculated with 100 ml of a preculture (LBmedium with 100 mg/l ampicillin) grown at 37° C. overnight.

The OD after the inoculation was 0.066. The aeration rate was adjustedto 2 l/min, the stirrer speed to 500 rpm and the temperature to 37° C.The pH was adjusted to pH 7.25 with 5 N NH₃ and kept constant throughoutthe fermentation.

After growth for 12 hours, the pO2 had fallen below 10% and most of theglucose had been consumed. After growth for 15 hours, the OD₆₀₀ was 14.2and the dry matter was 6.2 g/l. Feeding was started at this time. Thefeed solution consisted of: Glucose 700 g Magnesium sulphate × 7H₂O 28 gThiamine 0.5 g Tryptone 1.2 g Yeast extract 2.4 g Dist. water ad 1000 ml

The solution was sterilized by filtration. It was fed to the fermenterby a tubing pump. The pump delivery was controlled via the target pO₂,which was set at 20%. Control took place in such a way that the pump isswitched on at a pO₂ of >20%, and new glucose is fed in. The oxygenconsumption which starts up then causes the pO₂ to fall again. The pumpis switched off if the pO₂ falls below 20%. The stirrer speed andaeration rate were not changed. The OD₆₀₀ after growth for 64 hours was50.2. 5 litres of double concentrated TB medium were then fed into thefermenter, the temperature was reduced to 22° C., and the aeration ratewas set at 4 l/min.

The stirrer speed was raised to 700 rpm. The culture was then inducedwith 10 ml of anhydrotetracycline solution (2 mg/ml in DMF) for 6 hours.A sample was then taken to determine the activity. The activity assayrevealed a value of 223 U/ml of culture liquid. Over 220 mg of pureenzyme were obtained per ml of culture.

Example 9 Procedure for the Activity Assay

The measurements were carried out in an “Ultraspec 2000” photometer. Thefollowing solutions are employed for this purpose:

PIPES: 50 mM pH 6.5 incl. 2.2% Triton X-100

Glucose: 1 M in H₂O

PMS: 3 mM phenazine methosulphate in H₂O

NTB: 6.6 mM nitrotetrazolium blue in H₂O

CaCl₂: 1 M in H₂O

PQQ: 3 mM in DMSO

EDB *: 50 mM PIPES pH 6.5 incl. 0.1% Triton X-100, 1 mM CaCl₂, 0.1% BSA,6 μM PQQ.

EDB=enzyme dilution buffer

The working reagent consists of the following components:

25.5 ml of PIPES incl. 2.2% Triton X-100

0.9 ml of Glucose

2.0 ml of PMS

1.0 ml of NTB

The EDB and the working reagent should if possible be made up freshly.

Procedure for the activity determination:

Introduce 20 μl of the chosen sample dilution into microcuvettes (20 μlof EDB instead of the sample solution are added as zero value),

add 600 μl of working reagent and immediately start the measurement at570 nm for 3 minutes.

Calculation of the activity:

The change in absorption is measured as change in extinction/min; oneunit of GDH generates 0.5 μmol of formazan per minute. The followingformula applies:

U/ml=change in extinction/min×1.54×dilution factor ε=40 200 M⁻¹cm⁻¹

1. sPQQGDH of the sequence as depicted in FIG. 1, wherein the followingamino acids are exchanged by comparison with the wild type AcinetobacterLMD 79:41: position 21 has a N and 41 has an S and 47 has an L and 121has a V or an A and 149 has an A and 213 has an S and 244 has an I and320 has a G and 391 has an S and 452 has an S an d474 has an R and 480has a Q as amino acid.
 2. sPQQGDH of the sequences from claim 1, whereinadditionally the following amino acids may be exchanged by comparisonwith the wild type Acinetobacter LMD 79.41 (numbering as shown in FIG.1): there may be at positions 16 an H, at 18 an L, at 20 an F, at 40 aG, at 48 an I, at 61 an A, at 111 a T, at 154 a D, at 190 a D, at 293 anA, at 311 an S, at 314 an A, at 324 an L, at 333 an M, at 339 an S, at355 a G, at 366 a D, at 417 an N and at 418 an A.
 3. sPQQGDH of thesequences according to SEQ ID 3 to SEQ ID
 28. 4. sPQQGDH of thesequences from claim 1, wherein they are cloned into the vectorpASK-IBA3 and are heterologously expressed in a strain of the genusEscherichia.
 5. Process for preparing the sPQQGDH according to claims 1wherein a microorganism which expresses the sPQQGDH is cultivated, andthen the sPQQGDH is isolated.
 6. Reagent for detection of glucosecomprising one or more sPQQGDH enzymes according to claim
 1. 7. Sensorfor detecting glucose comprising one or more sPQQGDH enzymes accordingto claim
 1. 8. Method of using the plurality of sPQQGDH enzymesaccording to claims 1 for detecting glucose.
 9. sPQQGDH of the sequencesfrom claim 2, wherein they are cloned into the vector pASK-IBA3 and areheterologously expressed in a strain of the genus Escherichia. 10.sPQQGDH of the sequences from claim 3, wherein they are cloned into thevector pASK-IBA3 and are heterologously expressed in a strain of thegenus Escherichia.
 11. Process for preparing the sPQQGDH according toclaim 2, wherein a microorganism which expresses the sPQQGDH iscultivated, and then the sPQQGDH is isolated.
 12. Process for preparingthe sPQQGDH according to claim 3, wherein a microorganism whichexpresses the sPQQGDH is cultivated, and then the sPQQGDH is isolated.13. Reagent for detection of glucose comprising gone or more sPQQGDHenzymes according to claim
 2. 14. Reagent for detection of glucosecomprising gone or more sPQQGDH enzymes according to claim
 3. 15. Sensorfor detecting glucose comprising one or more sPQQGDH enzymes accordingto claim
 2. 16. Sensor for detecting glucose comprising one or moresPQQGDH enzymes according to claim
 3. 17. Method of using the pluralityof sPQQGDH enzymes according to claim 2 for detecting glucose. 18.Method of using the plurality of sPQQGDH enzymes according to claim 3for detecting glucose.